Resolution, configurational assignment, and enantiopharmacology of 2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol-4-yl]propionic acid, a potent GluR3- and GluR4-preferring AMPA receptor agonist

Abstract

We have previously shown that (RS)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol -4-yl] propionic acid (2-Me-Tet-AMPA) is a selective agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, markedly more potent than AMPA itself, whereas the isomeric compound 1-Me-Tet-AMPA is essentially inactive. We here report the enantiopharmacology of 2-Me-Tet-AMPA in radioligand binding and cortical wedge electrophysiological assay systems, and using cloned AMPA (GluR1-4) and kainic acid (KA) (GluR5, 6, and KA2) receptor subtypes expressed in Xenopus oocytes. 2-Me-Tet-AMPA was resolved using preparative chiral HPLC. Zwitterion (-)-2-Me-Tet-AMPA was assigned the (R)-configuration based on an X-ray crystallographic analysis supported by the elution order of (-)- and (+)-2-Me-Tet-AMPA using four different chiral HPLC columns and by circular dichroism spectra. None of the compounds tested showed detectable affinity for N-methyl-D-aspartic acid (NMDA) receptor sites, and (R)-2-Me-Tet-AMPA was essentially inactive in all of the test systems used. Whereas (S)-2-Me-Tet-AMPA showed low affinity (IC(50) = 11 microM) in the [(3)H]KA binding assay, it was significantly more potent (IC(50) = 0.009 microM) than AMPA (IC(50) = 0.039 microM) in the [(3)H]AMPA binding assay, and in agreement with these findings, (S)-2-Me-Tet-AMPA (EC(50) = 0.11 microM) was markedly more potent than AMPA (EC(50) = 3.5 microM) in the electrophysiological cortical wedge model. In contrast to AMPA, which showed comparable potencies (EC(50) = 1.3-3.5 microM) at receptors formed by the AMPA receptor subunits (GluR1-4) in Xenopus oocytes, more potent effects and a substantially higher degree of subunit selectivity were observed for (S)-2-Me-Tet-AMPA: GluR1o (EC(50) = 0.16 microM), GluR1o/GluR2i (EC(50) = 0.12 microM), GluR3o (EC(50) = 0.014 microM) and GluR4o (EC(50) = 0.009 microM). At the KA-preferring receptors GluR5 and GluR6/KA2, (S)-2-Me-Tet-AMPA showed much weaker agonist effects (EC(50) = 8.7 and 15.3 microM, respectively). It is concluded that (S)-2-Me-Tet-AMPA is a subunit-selective and highly potent AMPA receptor agonist and a potentially useful tool for studies of physiological AMPA receptor subtypes.

Tyr702 Is an Important Determinant of Agonist Binding and Domain Closure of the Ligand-Binding Core of GluR2

“…Desensitization of the responses was blocked by coapplication of a saturating concentration of cyclothiazide (100 M) in the presence of a saturating concentration (Ն 22-fold EC 50 ) of agonist. The EC 50 values of the agonists used have been reported previously (Banke et al, 1997;Coquelle et al, 2000;Kizelsztein et al, 2000;Vogensen et al, 2000;Campiani et al, 2001). The efficacies of KA and (S)-CPW399 relative to (S)-glutamate were measured at GluR2(Q) i :KA ϭ 0.212 Ϯ 0.032 (n ϭ 19), (S)-CPW399 ϭ 0.432 Ϯ 0.036 (n ϭ 21), and GluR3 i :KA ϭ 0.242 Ϯ 0.015 (n ϭ 21), (S)-CPW399 ϭ 0.147 Ϯ 0.017 (n ϭ 12).…”

Glutamate Receptor Ion Channels: Structure, Regulation, and Function

“…Data from X. laevis oocytes treated with con A (Brehm et al, 2003). Data from X. laevis oocytes (Vogensen et al, 2000). H͔kainate at recombinant kainate receptors (Lash et al, 2008).…”

“…At the same time, crystals are rarely formed on demand, low resolution can create ambiguities in protein threading and details of ligand binding pose, and dynamic aspects ranging from ligand interactions to domain coupling are not revealed by a static X-ray structure. Early in a project, in the absence of a three-dimensional structure, a homology model can offer unique insights, whereas in late phases of a project, possession of a crystal structure can benefit significantly from dynamic refinement and Stein et al (1992), Coquelle et al (2000), Nakanishi et al (1990), and Vogensen et al (2000). Coquelle et al (2000).…”

The Selective Activation of the Glutamate Receptor GluR5 by ATPA Is Controlled by Serine 741

“…AMPA activates the AMPA receptors expressed in oocytes with an EC 50 in the range of 1.3 to 3.5 M (Vogensen et al, 2000), whereas kainate receptors formed from the GluR5 to GluR7 subunits are either activated with EC 50 Ͼ 1 mM or not at all (Egebjerg et al, 1991;Sommer et al, 1992;Schiffer et al, 1997). Surprisingly, (S)-ATPA, an AMPA analog in which the methyl group at the 5-position in the isoxazole ring is replaced by a tert-butyl group (Lauridsen et al, 1985), exhibits a strong preference for GluR5 compared with the AMPA receptors (Clarke et al, 1997;Stensbøl et al, 1999).…”

“…These derivatives seem to contribute to the selectivity and potency both within the AMPA receptor family and be-tween the AMPA and kainate receptors. In particular, the AMPA analog with a 2-methyltetrazolyl substituent at the 5-position increases the potency and activates GluR4 and GluR1 with EC 50 values of 9 and 160 nM, respectively, but remains AMPA receptor-selective by activating the kainate receptor GluR5 with an EC 50 of 9 M (Vogensen et al, 2000). In contrast, the tert-butyl substitution in ATPA resulted in 100-fold higher potency at GluR5 (0.66 M) compared with GluR1 (62 M) when expressed in Xenopus laevis oocytes (Stensbøl et al, 1999).…”